In most eucaryotic genes, a common set of nucleotides has been found before the start site of RNA transcription. These nucleotides, referred to as either a TATA or Goldberg-Hogness box, are important determinants of transcription in vivo and in vitro. The control region for the late SV40 transcripts have been examined by site specific mutagenesis and by generation of deletion mutations. Mutations that enhance or suppress the transcriptional activity of a single start site have been identified and differ from the consensus Goldberg-Hogness box. When this upstream late transcription control region is converted to a sequence that is similar to the TATA box present in the SV40 early promoter, there is the strongest enhancement of late transcription in vitro. Spacing between a required GC rich domain and the TATA effects the level of early transcription suggesting that there is simultaneous contact in RNA polymerase II at these two sites.